The central goal of Project 2 is to understand the role of alternative splicing in cancer. Cancer cells display extensive qualitative and quantitative dysregulation of splicing, and a subset of the isoforms that are inappropriately expressed can contribute to tumorigenesis. The mechanisms and pathways through which the splicing-factor oncoprotein SRSF1 and related members of the SR protein family transform cells will be investigated. Organotypic culture, as well as orthotopic and transgenic mouse models will be used to study transformation promoted by these splicing factors and their cooperation with other oncogenes in different cancer contexts. How these factors themselves are regulated in normal cells and upregulated in cancer will be addressed. High-throughput methods will be employed to systematically identify the splicing targets of SR proteins in human cells, and selected targets involved in tumorigenesis will be analyzed in detail. Splicing factors that contribute to the distinctive glycolytic metabolism of cancer cells will be identified and characterized, and alternative splicing of pyruvate kinase pre-mRNA will be investigated as a potential therapeutic target by specifically manipulating this process in tumors.